JPS5935888B2 - Method for producing neem extract with cell division inhibiting activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for extracting a pharmacologically active ingredient having cytostatic activity from neem (Melia azadirachta).

Generally, in natural product chemistry, substances are extracted using low polar solvents such as benzene, ether, and ethyl acetate.
Many chemical structures have been determined for each component of a substance.

Recently, without using low polar solvents as in the past,
A method is used to obtain physiologically active substances by extracting natural products with a highly polar solvent such as water or hot water.

Examples of these include (A) Special Publication No. 52-28853;
(B) Special Publication No. 52-28854, (C) Special Publication No. 53-
No. 10124, (D) Japanese Patent Publication No. 53-10125, etc. are known.

In these examples, medicinal ingredients are suddenly extracted from the bark, leaves, flowers, fruits, branches, roots, and resin of neem (Melia azadirachta) using a hydrophilic solvent such as methyl alcohol or ethyl alcohol, water, hot water, etc. Regarding its medicinal efficacy, (B) is effective against acne, (B) is effective against rough skin, and (C) is effective against acne.
and (D) Staphylococcus aureus 20
It is stated that 9P has medicinal properties.

Generally, if the material is pretreated with a solvent with low polarity (low dielectric constant), undesired components can be removed in advance when extraction is performed with a solvent with high polarity (high dielectric constant).

This makes it possible to further reduce the amount of components other than the desired components in the desired extract, and to improve the medicinal efficacy.

Applying this principle, the present inventors have developed a pharmacological method that removes components other than the target components by selectively using solvents with different dielectric constants (polarity) for various parts of neem used in the above-mentioned known examples. The active ingredient was extracted and tested for cell division inhibiting activity using sea urchin eggs.

As a result, it was confirmed that the above-mentioned medicinal efficacy is particularly remarkable for the bark and leaves of neem, and that the method of the present invention increases the medicinal efficacy because there is a strong tendency to extract only the target components.

An object of the present invention is to provide a method for extracting pharmacologically active ingredients having cell division inhibiting activity from neem bark and leaves using solvents with different dielectric constants (polarity).

The present invention uses neem bark and/or leaves with a dielectric constant of 15 to 15.
Extraction treatment was performed with a hydrophilic solvent of 35, and the extraction residue was
Provided is a method for producing a neem extract having cell division inhibiting activity by extracting with 0'C water and further extracting the extraction residue with hot water.

The present invention is a method for subdividing medicinal ingredients based on the dielectric constant, and preferred examples of hydrophilic solvents with a dielectric constant of 15 to 35 include acetone, ethanol, and methanol.

If desired, neem bark and or leaves have a dielectric constant of 15.
-35, may be pretreated with an organic solvent having a dielectric constant of 10 or less, suitable examples of which include n-hexane, chloroform, benzene, and ethyl acetate.

The following table (■) shows the dielectric constants of these solvents. Next, the method for extracting the medicinal components of neem according to the present invention will be described in detail with reference to Examples.

[Example 1] 50ml of methanol was added to 5g of dried neem bark, and extraction was performed for one hour with occasional shaking.

Thereafter, this was passed through the mouth, and an additional 50 ml of methanol was added to the extraction residue, and the same operation was performed to repeat the extraction with methanol three times in total.

50 TLl of water (deionized water) at room temperature (approximately 25° C.) was added to the methanol extraction residue, and the same operation as above was repeated.

These two parts are called the water part. Further, 50 TLl of water was added to this water extraction residue, and the extraction operation was performed while heating in a boiling water bath, and the extraction operation was repeated three times in the same manner as above.

This is called the hot water fraction.

The water fraction and hot water fraction were dried and powdered using a rotary evaporator.

The dry powder has a water content of 203.6m9 and a hot water fraction of 180m9.
．． EMp was obtained.

[Example ■]

When 5'g of dried leaves of 2.~mu were subjected to the same extraction procedure as in Example 2, the water content was 1.3 Lii', the hot water fraction was 0.
A dry powder of sog was obtained.

[Example ■]

50 TLl of benzene was added to the dried neem bark 59, and extraction was performed for one hour with occasional shaking.

Thereafter, this was passed through the mouth, and an additional 50 ml of benzene was added to the extraction residue and the same operation was carried out, repeating the extraction with benzene three times in total.

Next, 50 ml of ethanol was added to the benzene extraction residue, and the same operation as that for benzene was repeated.

50rrLl of water (deionized water) at room temperature (approximately 25°C) was added to the ethanol extraction residue, and the same operation as above was repeated.

These two fractions are referred to as the water fraction.

Furthermore, 50 ml of water was added to this water extraction residue, and the extraction operation was performed while heating in a boiling water bath.
The extraction operation was repeated twice.

This is called the hot water fraction.

The above water fraction and hot water fraction were dried and powdered using a rotary evaporator.

The dry powder has a water content of 235.4% and a hot water fraction of 178.
67n? Obtained.

The water used in the above examples is deionized water, but is not limited to this, and various types of water can be used, such as tap water, well water, salt water, distilled water, RO water (water obtained using a reverse osmosis membrane), etc. Can be used.

The temperature of the water used is preferably 0 to 40°C.

〔test〕

The cell division inhibiting activity of the above-mentioned neem extract obtained by the method of the present invention was measured in the following manner using sea urchin eggs.

200 to 300 fertilized eggs of Bafun sea urchin or purple sea urchin were placed in each test tube along with 2 ml of seawater.

Almost simultaneously, the sample solution (aqueous solution or DMSO (Dimethlsulfoxide) solution) obtained in the above example was added to 0.
2 mrrtm13 was added.

After this, the state of cell division in the fertilized sea urchin eggs was observed under a microscope.

The results are shown in Table ■ below. In Table ■, those that prevent egg cell division are ++10, those that prevent 50 to 80% of egg division are +10, and those that allow egg cell division up to 1 to 2 times are *,
Those that inhibit progression beyond the blastula stage are indicated as △, and those that do not inhibit division are indicated as -.

From the test results in Table 1 above, it can be seen that the hot water fraction sample obtained by the method of the present invention has more than 10 times the medicinal efficacy than the water fraction sample, and this is particularly noticeable in the bark of neem than in the leaves. It has been proven to have medicinal properties.

The need to perform extraction and fractionation using a variety of solvents with a low dielectric constant corresponds to the need to exclude components other than the intended components.

The test results revealed that the neem extract obtained by the method of the present invention effectively inhibited cell division at a lower concentration, that is, a higher concentration of pharmacologically active ingredients was extracted.

As described above, it is expected that the medicinal ingredients obtained by the method of the present invention, which can very effectively inhibit cell division, will find applications in a wide range of fields such as concentrated medicines and pharmaceuticals.

The formulation, effective dosage, detoxification, etc. are determined within the limits permitted in the field of application.

[Comparative example]

The IR (infrared rays) absorption spectrum of the neem extract obtained by the method of the present invention was compared with that of the known example mentioned at the beginning.

Figure 1 shows the IR absorption spectra of water extracted components of neem bark, and shows the known examples (A), (13) or (C).
).

FIG. 2 shows the IR absorption and absorption spectra of hot water extracted components of neem bark and leaves, and is similar to (C) of the above-mentioned known example.

FIG. 3 shows the water content I in the above-mentioned embodiment
FIG. 4 shows the IR absorption and absorption spectrum of the hydrothermal fraction of the above-mentioned Example (2) of the present invention.

When comparing the above IR absorption and absorption spectra in the same figure, it is found that based on the embodiment of the present invention shown in FIG. It is presumed that this is linked to the medicinal efficacy that is clearly shown in .

The above-mentioned tests clearly demonstrate that the neem extract obtained by the method of the present invention has a marked difference in medicinal efficacy compared to the neem extract obtained by the method described in the known example.

This is because the method of the present invention achieves a purification process that eliminates components other than the target components as much as possible, and this is not seen at all in conventional methods, and it is also seen in the infrared absorption spectrum diagram. The specificity is clearly expressed.

Purification of medicinal ingredients is often difficult, but the present invention can be carried out through a relatively simple process, and the fact that the medicinal efficacy of the obtained extract is significantly increased indicates the effectiveness of the present invention. There is something remarkable about this that is not seen in conventional examples.

A wide variety of applications are expected to be developed in the fields of medicine, agricultural chemicals, etc., and the active ingredients can be appropriately reprinted using appropriate solvents for each field.

The present invention is not limited to what has been described above, and various modifications can be made within the scope of the present invention.

For example, similar effects have been obtained not only with the combinations of solutions used for extraction as shown in Examples, but also with other combinations of solvents exemplified in Table I.

[Brief explanation of drawings]

Figure 1 is the IR absorption and absorption spectrum of the water extract from neem bark. Figure 2 is the IR absorption spectrum of the water extract from neem leaves. Figure 3 is the water extract from neem bark obtained by the method of the present invention. Figure 4 shows the IR absorption spectrum of the hot water extracted components of neem bark obtained by the method of the present invention.

Claims (1)

[Claims] 1 Neem (Melia azadirachta) bark and/or leaves with a dielectric constant of 15
Extraction treatment with ~35 hydrophilic solvent and the extraction residue at 0℃
A method for producing a neem extract having cell division inhibiting activity, which comprises extracting with water at ~40°C and further extracting the extraction residue with hot water. 2. The method for producing a neem extract having cell division inhibiting activity according to claim 1, wherein the hydrophilic solvent having a dielectric constant of 15 to 35 is methanol. 3. The method for producing a neem extract having cell division inhibiting activity according to claim 1, wherein the hydrophilic solvent having a dielectric constant of 15 to 35 is ethanol. 4. Has cell division inhibiting activity according to claims 1 to 3, characterized in that it is extracted with an organic solvent with a dielectric constant of 10 or less before being treated with a hydrophilic solvent with a dielectric constant of 15 to 35. Method for producing neem extract.